Nucleic acid probe technology has developed rapidly in recent years as researchers have discovered its value for detection of various diseases, organisms or genetic features which are present in very small quantities in a test sample. The use of probes is based upon the concept of complementarity. In DNA the two strands are bound to each other by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions which disrupt the hydrogen bonding. The released single strands will reassociate only with another strand having a complementary sequence of nucleotides. This hybridization process can occur in solution or on a solid substrate. RNA is usually single-stranded. It can also hybridize with another strand or portion thereof which has a complementary sequence of nucleotides.
A target nucleic acid sequence of DNA or RNA of a target organism or cell may be only a small portion of the total strand, so that it is very difficult to detect its presence using most known labeled DNA probes. Much research has been carried out to overcome this problem including improvements in probe sensitivity and synthesis of nucleic acids.
A significant advance in the art is described in U.S. Pat. Nos. 4,683,195 (issued Jul. 28, 1987 to Mullis et al) and 4,683,202 (issued Jul. 28, 1987 to Mullis). Without going into extensive detail, these patents describe an amplification method wherein primers are hybridized to nucleic acid templates in the presence of a polymerization agent (such as a polymerase) and four nucleotide triphosphates, and extension products are formed from the primers. These products are denatured and used as templates in a cycling reaction which amplifies the number and amount of existing nucleic acids to facilitate their subsequent detection. The amplification method can be carried out cyclically as many times as desired to produce a larger quantity of detectable material from a small amount of target nucleic acid sequence.
In the amplification method described above, two primers are used for the target nucleic acid to be amplified. Design of an efficient diagnostic assay based upon the polymerase chain reaction depends greatly on the efficiency with which the primers hybridize with the target nucleic acid. In the best case for amplification, the nucleic acid sequence to be amplified is completely complementary with the primer, at least near the 3' end of the target sequence where extension will occur. Thus, only one primer per strand is needed for effective amplification. It is known from the art that where the target sequence is not entirely known, at least at the 3' end, a collection of primers having all possible codon variations can be used in order to have at least one primer which is completely complementary.
The use of a collection of primers may be used to accomplish the amplification process desired, but it is impractical due to its expense and may be inefficient or ineffective in many instances. The preparation of the collection of random primers is wasteful and leads to the use of competitive non-extending primers. Moreover, the greater the uncertainty of the target nucleotide sequence, the collection of primers needed is greatly enlarged.
Viral genomes, particularly those of RNA viruses and retroviruses, contain multiple base alterations, additions, duplications and deletions. The variability of these viruses has been attributed to the low fidelity and lack of proofreading functions of polymerases responsible for replication (see Steinhauer et al, Ann. Rev. Microbiol., 41, pp. 409-433, 1986). Repeated rounds of infection further increase variability. The effects of such variations in the natural history of infection of a given virus is only beginning to be understood.
Thus, in the detection of heterogeneous DNA from retroviruses, the target nucleic acid is highly variable, and complete identity is not always known. With HIV-I, for example, a variety of sequences in the genome produces a viable virus. Base substitutions are known to occur at random and frequent intervals over the entire genome. Thus, different isolates are likely to have viral DNA which have different nucleic acid sequences which could lead to mismatches with primers thought to be complementary.
Such mismatches will considerably reduce the efficiency of amplification by primers, especially when the mismatch between target and primer occurs at or near the 3' end of the primer. In other words, mismatches lead to a slowing down of the amplification process because the kinetics of priming and primer extension are changed (see for example, Tinoco, Jr., Proc. Nat. Acad. Sci.(USA), 85, 6252, 1988). In the worst case, no amplification will occur as the primer fails to attach to the target, or if it attaches, formation of an extension product is inhibited (that is, the primer "misfires").
It would be desirable to have an efficient means for amplifying and detecting nucleic acids even if there is a mismatch between a targeted sequence of the nucleic acid and a primer at or near the 3' end of the primer.